U.S. patent application number 09/543385 was filed with the patent office on 2002-06-27 for method for treating restenosis with a2a adenosine receptor agonists.
Invention is credited to Linden, Joel M, Sarembock, Ian, Sheld, W Michael, Sullivan, Gail W.
Application Number | 20020082240 09/543385 |
Document ID | / |
Family ID | 26672386 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082240 |
Kind Code |
A1 |
Linden, Joel M ; et
al. |
June 27, 2002 |
METHOD FOR TREATING RESTENOSIS WITH A2A ADENOSINE RECEPTOR
AGONISTS
Abstract
Agonists of A.sub.2A adenosine receptors in combination with
rolipram, its derivatives or other Type IV phosphodiesterase (PDE)
inhibitors are effective for the treatment of inflammatory
diseases.
Inventors: |
Linden, Joel M;
(Charlottseville, VA) ; Sullivan, Gail W;
(Charlottesville, VA) ; Sarembock, Ian;
(Charlottesville, VA) ; Sheld, W Michael;
(Earlysville, VA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Family ID: |
26672386 |
Appl. No.: |
09/543385 |
Filed: |
April 4, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09543385 |
Apr 4, 2000 |
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09003930 |
Jan 8, 1998 |
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09003930 |
Jan 8, 1998 |
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08272821 |
Jul 11, 1994 |
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5877180 |
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Current U.S.
Class: |
514/46 |
Current CPC
Class: |
A61K 31/522 20130101;
A61K 31/4015 20130101; Y02A 50/30 20180101; A61K 31/50 20130101;
A61K 31/7076 20130101; A61K 45/06 20130101; Y02A 50/411 20180101;
A61K 31/52 20130101; A61K 31/52 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/46 |
International
Class: |
A61K 031/70 |
Goverment Interests
[0002] The present invention was made with the assistance of U.S.
Government funding (NIH Grant R01-HL 37942). The U.S. Government
may have some rights in this invention.
Claims
What is claimed:
1. A method of treating inflammatory disease, comprising
administering to a patient in need thereof an agonist of an
A.sub.2A adenosine receptor in combination with rolipram, a
rolipram derivative or other compound that is a Type IV
phosphodiesterase inhibitor.
2. The method of claim 1, wherein said disease is selected from the
group consisting of: autoimmune diseases (lupus erythenatosis),
multiple sclerosis, type I diabetes mellits, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, osteoporosis,
arthritis, allergic diseases (asthma), infectious diseases
(sepsis),septic shock, infectious arthritis, endotoxic shock, gram
negative shock, toxic shock,cerebral malaria, bacterial meningitis,
adult respiratory distress syndrome, TNF.alpha.-enhanced HIV
replication and TNF.alpha. inhibition of reverse transciptase
inhibitor activity, wasting diseases (cachexia secondary to cancer
and HIV), skin diseases (psoriasis), contact dermatitis, eczema,
infectious skin ulcers, cellulitis,organ transplant rejection,
graft versus host disease, adverse effects from amphotericin B
treatment, adverse effects from interleukin-2 treatment, adverse
effects from OKT3 treatment, adverse effects from GM-CSF treatment,
adverse effects of cyclosporine treatment and adverse effects of
aminoglycoside treatment, ischemia, mucositis, infertility from
endometriosis, atherosclerosis, peripheral vascular disease,
restenosis following angioplasty, inflammatory aortic aneurysm,
ischemia/reperfusion damage, vasculitis, stroke, congestive heart
failure, hemorrhagic shock, vasospasm following subarachnoid
hemorrhage, vasospasm following cerebrovascular accident,
pleauritis, pericarditis, and encephalitis.
3. The method of claim 1, wherein said agonist of an A.sub.2A
adenosine receptor has the formula (I) 9wherein X is a group
selected from the group consisting of --OR.sup.1,
--NR.sup.2R.sup.3, and --NH--N.dbd.R.sup.4; wherein R.sup.1 is
C.sub.1-4-alkyl; C.sub.1-4-alkyl substituted with one or more
C.sub.1-4-alkoxy groups, halogens (fluorine, chlorine, or bromine),
hydroxy groups, amino groups, mono(C.sub.1-4-alkyl)amino groups,
di(C.sub.1-4-alkyl)amino groups, or C.sub.6-10-aryl groups (wherein
the aryl groups may be substituted with one or more halogens
(fluorine, chlorine, or bromine), C.sub.1-4-alkyl groups, hydroxy
groups, amino groups, mono(C.sub.1-4-alkyl)amino groups, or
di(C.sub.1-4alkyl)amino groups); C.sub.6-10-aryl; or
C.sub.6-10-aryl substituted with one or more halogens (fluorine,
chlorine, or bromine), hydroxy groups, amino groups,
mono(C.sub.1-4-alkyl)amino groups, or di(C.sub.1-4 alkyl)amino
groups, or C.sub.1-4-alkyl groups; one of R.sup.2 and R.sup.3 has
the same meaning as R.sup.1 and the other is hydrogen; R.sup.4 is a
group having the formula 10wherein each of R.sup.5 and R.sup.6
independently may be hydrogen, C.sub.3-7-cycloalkyl, or any of the
meanings of R.sup.1, provided that R.sup.5 and R.sup.6 are not both
hydrogen; or a pharmaceutically acceptable salt thereof.
4. The method of claim 1, wherein said agonist of an A.sub.2A
adenosine receptor is selected from the group consisting of: 11
5. The method of claim 1, wherein said Type IV phosphodiesterase
inhibitor is a compound having formula (V): 12wherein R.sup.18 and
R.sup.19 each are alike or different and are hydrocarbon radicals
having up to 18 carbon atoms with at least one being other than
methyl, a heterocyclic ring, or alkyl of 1-5 carbon atoms which is
substituted by one or more of halogen atoms, hydroxy, carboxy,
alkoxy, alkoxycarbonyl or an amino group; or amino.
6. The method of claim 1, wherein said type IV phosphodiesterase
inhibitor is rolipram.
7. The method of claim 1, wherein said agonist of an A.sub.2A
adenosine receptor is 13and said Type IV phosphosterase inhibitor
is rolipram.
8. The method of claim 1, wherein said agonist of an A.sub.2A
adenosine receptor is 14
9. The method of claim 1, wherein said A.sub.2A adenosine receptor
agonist and said Type IV phosphosterase inhibitor are
coadministered together to the patient in need thereof.
10. A pharmaceutical composition comprising an effective amount of
an agonist of an A.sub.2A adenosine receptor in combination with
rolipram or a rolipram derivative or a rolipram analogue or other
Type IV phosphodiesterase inhibitors.
11. The pharmaceutical composition of claim 10, wherein said
agonist of an A.sub.2A adenosine receptor has the formula (I)
15wherein X is a group selected from the group consisting of
--OR.sup.1, --NR.sup.2R.sup.3, and --NH--N.dbd.R.sup.4; wherein
R.sup.1 is C.sub.1-4-alkyl; C.sub.1-4-alkyl substituted with one or
more C.sub.1-4-alkoxy groups, halogens (fluorine, chlorine, or
bromine), hydroxy groups, amino groups, mono(C.sub.1-4-alkyl)amino
groups, di(C.sub.1-4-alkyl)amino groups, or C.sub.6-10-aryl groups
(wherein the aryl groups may be substituted with one or more
halogens (fluorine, chlorine, or bromine), C.sub.1-4-alkyl groups,
hydroxy groups, amino groups, mono(C.sub.1-4-alkyl)amino groups, or
di(C.sub.1-4alkyl)amino groups); C.sub.6-10-aryl; or
C.sub.6-10-aryl substituted with one or more halogens (fluorine,
chlorine, or bromine), hydroxy groups, amino groups,
mono(C.sub.1-4-alkyl)amino groups, or di(C.sub.1-4 alkyl)amino
groups, or C.sub.1-4-alkyl groups; one of R.sup.2 and R.sup.3 has
the same meaning as R.sup.1 and the other is hydrogen; R.sup.4 is a
group having the formula 16wherein each of R.sup.5 and R.sup.6
independently may be hydrogen, C.sub.3-7-cycloalkyl, or any of the
meanings of R.sup.1, provided that R.sup.5 and R.sup.6 are not both
hydrogen; or a pharmaceutically acceptable salt thereof.
12. The pharmaceutical composition of claim 10, wherein said
agonist of an A.sub.2A adenosine receptor is selected from the
group consisting of 17
13. The pharmaceutical composition of claim 10, wherein said Type
IV phosphodiesterase inhibitor is a compound having formula (V):
18wherein R.sup.18 and R.sup.19 each are alike or different and are
hydrocarbon radicals having up to 18 carbon atoms with at least one
being other than methyl, a heterocyclic ring, or alkyl of 1-5
carbon atoms which is substituted by one or more of halogen atoms,
hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino group; amino;
R' is a hydrogen atom, alkyl, aryl or acyl; and X is an oxygen atom
or a sulfur atom.
14. The pharmaceutical composition of claim 10, wherein said type
IV phosphodiesterase inhibitor is rolipram.
15. The pharmaceutical composition of claim 10, wherein said
agonist of an A.sub.2A adenosine receptor is 19and said Type IV
phosphosterase inhibitor is rolipram.
16. The pharmaceutical composition of claim 10, wherein said
agonist of an A.sub.2A adenosine receptor is 20and said Type IV
phosphosterase inhibitor is rolipram.
17. The method of claim 1, further comprising administering said
agonist of an A.sub.2A adenosine receptor in combination with
rolipram, a rolipram derivative or other compound that is a Type IV
phosphodiesterase inhibitor during and for a limited time after
balloon angioplasty to reduce frequence and extent of
restenosis.
18. The method of claim 1, further comprising administering said
agonist of an A.sub.2A adenosine receptor in combination with
rolipram, a rolipram derivative or other compound that is a Type IV
phosphodiesterase inhibitor in conjunction with a gene delivery
modality to limit inflammation and thereby improve efficiency and
stability of gene therapy.
19. The method of claim 1, wherein said gene delivery modality is
selected from the group comprising viruses and lipid vesicles.
Description
RELATED APPLICATION
[0001] This application is a continuation-in part of co-pending
U.S. patent application Ser. No. 08/272,821, filed Jul. 22, 1994 to
Linden et al., which is incorporated herein in its entirety by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods and compositions
for treating inflammatory diseases.
[0005] 2. Discussion of the Background
[0006] The release of inflammatory cytokines such as tumor necrosis
factor-alpha (TNF.alpha.) by leukocytes is a means by which the
immune system combats pathogenic invasions, including infections.
Cytokines stimulate neutrophils to enhance oxidative (e.g.,
superoxide and secondary products) and nonoxidative (e.g.,
myeloperoxidase and other enzymes) inflammatory activity.
Inappropriate and over-release of cytokines can produce
counterproductive exaggerated pathogenic effects through the
release of tissue damaging oxidative and nonoxidative products
(Tracey, K. G., et al., J. Exp. Med., vol. 167, pp. 1211-1227
(1988); and Mnnel, D. N., et al., Rev. Infect. Dis., vol. 9 (suppl
5), pp. S602-S606 (1987)).
[0007] For example, inflammatory cytokines have been shown to be
pathogenic in: arthritis (Dinarello, C. A., Semin. Immunol., vol.
4, pp. 133-45 (1992)); ischemia (Seekamp, A., et al.,
Agents-Actions-Supp., vol. 41, pp. 137-52 (1993)); septic shock
(Mnnel, D. N., et al., Rev. Infect. Dis., vol. 9, (suppl 5), pp.
S602-S606 (1987)); asthma (Cembrzynska Nowak M., et al., Am. Rev.
Respir. Dis., vol. 147, pp. 291-5 (1993)); organ transplant
rejection (Imagawa, D. K., et al., Transplantation, vol. 51, pp.
57-62 (1991)); multiple sclerosis (Hartung, H. P., Ann. Neurol.,
vol. 33, pp. 591-6 (1993)); and AIDS (Matsuyama, T., et al., AIDS,
vol. 5, pp. 1405-1417 (1991)). In addition, superoxide formation in
leukocytes has been implicated in promoting replication of the
human immunodeficiency virus (HIV) (Legrand-Poels, S., et al., AIDS
Res. Hum. Retroviruses, vol. 6, pp. 1389-1397 (1990)).
[0008] It is well known that adenosine and some relatively
nonspecific analogs of adenosine decrease neutrophil production of
inflammatory oxidative products (Cronstein, B. N., et al., Ann.
N.Y. Acad. Sci., vol. 451, pp. 291-314 (1985); Roberts, P. A., et
al., Biochem. J., vol. 227, pp. 66.9-674 (19-85); Schrier, D. J.,
et al., J. Immunol., vol. 137, pp. 3284-3289 (1986); Cronstein, B.
N., et al., Clinical Immunol. and Immunopath., vol. 42, pp. 76-85
(1987); Iannone, M. A., et al., in Topics and Perspectives in
Adenosine Research, E. Gerlach et al., eds., Springer-Verlag,
Berlin, pp. 286-298 (1987); McGarrity, S. T., et al., J. Leukocyte
Biol., vol. 44, pp. 411421 (1988); De La Harpe, J., et al., J.
Immunol., vol. 143, pp. 596-602 (1989); McGarrity, S. T., et al.,
J. Immunol., vol. 142, pp. 1986-1994 (1989); and Nielson, C. P., et
al., Br. J.Pharmacol., vol. 97, pp. 882-888 (1989)). For example,
adenosine has been shown to inhibit superoxide release from
neutrophils stimulated by chemoattractants such as the synthetic
mimic of bacterial peptides, f-met-leu-phe (fMLP), and the
complement component C.sub.5a (Cronstein, B. N.,et al., J. Immunol,
vol. 135, pp. 1366-1371 (1985)). Adenosine can decrease the greatly
enhanced oxidative burst of PMN (neutrophil) first primed with
TNF-.alpha. (an inflammatory cytokine) and then stimulated by a
second stimulus such as f-met-leu-phe (Sullivan, G. W., et al.,
Clin. Res., vol. 41, p. 172A (1993)). There is evidence that in
vivo adenosine has anti-inflammatory activity (Firestein, G. S., et
al., Clin. Res., vol. 41, p. 170A (1993); and Cronstein, B. N., et
al., Clin. Res., vol. 41, p. 244A (1993)). Additionally, it has
been reported that adenosine can decrease the rate of HIV
replication in a T-cell line (Sipka, S., et al., Acta. Biochim.
Biopys. Hung., vol. 23, pp. 75-82 (1988)).
[0009] It has been suggested that there is more than one subtype of
adenosine receptor on neutrophils that have opposite effects on
superoxide release (Cronstein, B. N., et al., J. Clin. Invest.,
vol. 85, pp. 1150-1157 (1990)). The existence of A.sub.2A receptor
on neutrophils was originally demonstrated by Van Calker et al.
(Van Calker, D., et al., Eur. J. Pharmacology, vol. 206, pp.
285-290 (1991)).
[0010] There has been progressive development of compounds that are
more and more potent and selective as agonists of A.sub.2A
adenosine receptors based on radioligand binding assays and
physiological responses. Initially, compounds with little or no
selectivity for A.sub.2A receptors were used, such as adenosine
itself or 5'-carboxamides of adenosine, such as
5'-N-ethylcarboxamidoadenosine (NECA) (Cronstein, B. N., et al., J.
Immunol., vol. 135, pp. 1366-1371 (1985)). Later, it was shown that
addition of 2-alkylamino substituents increased potency and
selectivity, e.g. CV1808 and CGS21680 (Jarvis, M. F., et al., J.
Pharmacol. Exp. Ther., vol. 251, pp. 888-893 (1989)).
2-Alkoxy-substituted adenosine derivatives such as WRC-0090 are
even more potent and selective as agonists on the coronary artery
A.sub.2A receptor (Ukena, M., et al., J. Med. Chem., vol. 34, pp.
1334-1339 (1991)). The 2-alkylhydrazino adenosine derivatives, e.g.
SHA 211 (also called WRC-0474) have also been evaluated as agonists
at the coronary artery A.sub.2A receptor (Niiya, K., et al., J.
Med. Chem., vol. 35, pp. 45574561(1992)).
[0011] There is one report of the combination of relatively
nonspecific adenosine analogs, R-phenylisopropyladenosine (R-PIA)
and 2-chloroadenosine (Cl-Ado) with a phosphodiesterase (PDE)
inhibitor resulting in a lowering of neutrophil oxidative activity
(Iannone, M. A., et al., in Topics and Perspectives in Adenosine
Research, E. Gerlach et al., Eds., Springer-Verlag, Berlin, pp.
286-298 (1987)). However, R-PIA and Cl-Ado analogs are actually
more potent activators of A.sub.1 adenosine receptors than of
A.sub.2A adenosine receptors and, thus, are likely to cause side
effects due to activation of A.sub.1 receptors on cardiac muscle
and other tissues causing effects such as "heart block".
[0012] Linden et al. Ser. No. 08/272,821 is based on the discovery
that inflammatory diseases may be effectively treated by the
administration of drugs which are selective agonists of A.sub.2A
adenosine receptors, preferably in combination with a
phosphodiesterase inhibitor. An embodiment of the Linden et al.
invention provides a method for treating inflammatory diseases by
administering an effective amount of an A.sub.2A adenosine receptor
of the following formula: 1
[0013] wherein X is a group selected from the group consisting of
--OR.sup.1, --NR.sup.2R.sup.3, and --NH--N.dbd.R.sup.4;
[0014] wherein R.sup.1 is C.sub.1-4-alkyl; C.sub.1-4-alkyl
substituted with one or more C.sub.1-4-alkoxy groups, halogens
(-fluorine, chlorine, or bromine), hydroxy groups, amino groups,
mono(C.sub.1-4-alkyl)amino groups, di(C.sub.1-4,-alkyl)amino
groups, or C.sub.6-10-aryl groups (wherein the aryl groups may be
substituted with one or more halogens (fluorine, chlorine, or
bromine), C.sub.1-4-alkyl groups, hydroxy groups, amino groups,
mono(C.sub.1-4-alkyl)amino groups, or di(C.sub.1-4 alkyl)amino
groups); C.sub.6-10-aryl; or C.sub.6-10-aryl substituted with one
or more halogens (fluorine, chlorine, or bromine), hydroxy groups,
amino groups, mono(C.sub.1-4-alkyl)amino groups, or di(C.sub.1-4
alkyl)amino groups, or C.sub.1-4-alkyl groups;
[0015] one of R.sup.2 and R.sup.3 has the same meaning as R.sup.1
and the other is hydrogen;
[0016] R.sup.4 is a group having the formula: 2
[0017] wherein each of R.sup.5 and R.sup.6 independently may be
hydrogen, C.sub.3-7-cycloalkyl, or any of the meanings of R.sup.1,
provided that R.sup.5 and R.sup.6 are not both hydrogen; and
[0018] R is --CH.sub.2OH, --CH.sub.2H, --CO.sub.2R.sup.7, or
--C(.dbd.O)NR.sup.8R.sup.9; wherein R.sup.7 has the same meaning as
R.sup.1 and wherein R.sup.8 and R.sup.9 have the same meanings as
R.sup.5 and R.sup.6 and R.sup.8 and R.sup.9 may both be
hydrogen.
[0019] In a preferred embodiment, the Linden et al. invention
involves the administration of a Type IV phosphodiesterase (PDE)
inhibitor in combination with the A.sub.2A adenosine receptor
agonist. The Type IV phosphodiesterase (PDE) inhibitor can be
racemic and optically active 4-(polyalkoxyphenyl)-2-pyrrolidones of
the following formula: 3
[0020] (disclosed and described in U.S. Pat. No. 4,193,926) wherein
R.sup.18 and R.sup.19 each are alike or different and are
hydrocarbon radicals having up to 18 carbon atoms with at least one
being other than methyl, a heterocyclic ring, or alkyl of 1-5
carbon atoms which is substituted by one or more of halogen atoms,
hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino group; amino;
R' is a hydrogen atom, alkyl, aryl or acyl; and X is an oxygen atom
or a sulfur atom.
[0021] Rolipram is an example of a suitable Type IV
phosphodiesterase or PDE inhibitor included within the above
formula. Rolipram has the following structure: 4
[0022] The present invention is based on the inventors' discovery
that improved effective treatment of inflammatory disease is
achieved by the administration of certain agonists of A.sub.2A
adenosine receptors in combination with rolipram or rolipram
derivatives that are Type IV phosphodiesterase or PDE
inhibitors.
SUMMARY OF THE INVENTION
[0023] Accordingly, one object of the present invention is to
provide a novel and improved method for treating inflammatory
diseases.
[0024] It is another object of the present invention to provide
novel and improved compositions for the treatment of inflammatory
disease.
[0025] These and other objects, which will become better understood
during the course of the following detailed description, have been
achieved by the inventors' discovery of improved compositions and
methods for effectively treating inflammatory diseases by
administration of an agonist of an A.sub.2A adenosine receptor in
combination with rolipram or a rolipram derivative that is a Type
IV phosphodiesterase (PDE) inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0027] FIG. 1 illustrates the relative potencies of adenosine
analogs to modulate TNF.sub..alpha.-primed fMLP-stimulated
polymorphonuclear cell (PMN) chemiluminescence as a measure of PMN
production of oxidative products (0, no TNF.alpha.; .DELTA.,
WRC-0474[SHA 211]+TNF.alpha.; .quadrature., CGS 21680+TNF.alpha.;
and .tangle-solidup., adenosine+TNF.alpha.);
[0028] FIG. 2 illustrates the synergistic effect of WRC-0474[SHA
211] and 4-(3cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone
(rolipram) in inhibiting TNF.alpha.-primed (10 U/ml),
fMLP-stimulated (100 nM) PMN superoxide production: 0, no
4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrro- lidone;
.tangle-solidup., 3 nM 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrr-
olidone; .quadrature., 30 nM
4-(3-cyclopentyloxy4-methoxyphenyl)-2-pyrroli- done; and, 300 nM
4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone.
[0029] FIG. 3 illustrates the synergistic effect of WRC-0474[SHA
211] and rolipram in inhibiting TNF.alpha.-stimulated adherent PMN
superoxide release;
[0030] FIG. 4 illustrates the effect of WRC-0474[SHA 211] and
rolipram on TNF.alpha.-stimulated PMN adherence to a fibrinogen
coated surface;
[0031] FIG. 5 illustrates synergy between A.sub.2A adenosine
receptor agonists and Rolipram in inhibiting superoxide release
from TNF.alpha.-stimulated adherent human neutrophils;
[0032] FIG. 6 illustrates the effects of WRC-0470 and rolipram on
the oxidative activity of neutrophils in whole blood;
[0033] FIG. 7 illustrates the effects of WRC-0470 and rolipram on
the release of TNF.alpha. from adherent human monocytes and that
this activity is dependent on binding of the adenosine agonist to
A.sub.2A adenosine receptors.
[0034] FIG. 8 illustrates the effect of WRC-0470 on white blood
cell pleocytosis in rats.
[0035] FIG. 9 illustrates the effect of WRC-0470 on
blood-brain-barrier permeability in rats;
[0036] FIG. 10 illustrates the effect of rolipram on white blood
cell pleocytosis in rats; and
[0037] FIG. 11 illustrates the combined effect of WRC-0470 and
rolipram on white blood cell pleocytosis in rats.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] Thus, in a first embodiment, the present invention provides
a method for treating inflammatory diseases by administering an
effective amount of a compound of formula (I): 5
[0039] wherein X is a group selected from the group consisting of
--OR.sup.1, --NR.sup.2R.sup.3, and --NH--N.dbd.R.sup.4;
[0040] wherein R.sup.1 is C.sub.1-4-alkyl; C.sub.1-4-alkyl
substituted with one or more C.sub.1-4-alkoxy groups, halogens
(-fluorine, chlorine, or bromine), hydroxy groups, amino groups,
mono(C.sub.1-4-alkyl)amino groups, di(C.sub.1-4-alkyl)amino groups,
or C.sub.6-10-aryl groups (wherein the aryl groups may be
substituted with one or more halogens (fluorine, chlorine, or
bromine), C.sub.1-4-alkyl groups, hydroxy groups, amino groups,
mono(C.sub.1-4-alkyl)amino groups, or di(C.sub.1-4 alkyl)amino
groups); C.sub.6-10-aryl; or C.sub.6-10-aryl substituted with one
or more halogens (fluorine, chlorine, or bromine), hydroxy groups,
amino groups, mono(C.sub.1-4-alkyl)amino groups, or di(C.sub.1-4
alkyl)amino groups, or C.sub.1-4-alkyl groups;
[0041] one of R.sup.2 and R.sup.3 has the same meaning as R.sup.1
and the other is hydrogen;
[0042] R.sup.4 is a group having the formula (II) 6
[0043] wherein each of R.sup.5 and R.sup.6 independently may be
hydrogen, C.sub.3-7-cycloalkyl, or any of the meanings of R.sup.1 ,
provided that R.sup.5 and R.sup.6 are not both hydrogen;
[0044] Examples of suitable C.sub.6-10-aryl groups include phenyl
and naphthyl.
[0045] Preferably the compound of formula (I) has X being a group
of the formula (III)
--O--CH.sub.2.paren close-st..sub.n--Ar (III)
[0046] wherein n is an integer from 1-4, preferably 2, and Ar is a
phenyl group, tolyl group, naphthyl group, xylyl group, or mesityl
group. Most preferably Ar is a para-tolyl group and n=2.
[0047] Even more preferably, the compound of formula (IV) has X
being a group of the formula (I)
--NH--N.dbd.CHCy (IV)
[0048] wherein Cy is a C.sub.3-7-cycloalkyl group, preferably
cyclohexyl or a C.sub.1-4 alkyl group, preferably isopropyl.
[0049] Specific examples of such compounds of formula (I) include
WRC-0470, WRC-0474 [SHA 211], WRC-0090 and WRC-0018, shown below:
7
[0050] Of these specific examples, WRC-0474[SHA 211] and WRC-0470
are particularly preferred.
[0051] Such compounds may be synthesized as described in:
Hutchinson, A. J., et al., J. Pharmacol. ExD. Ther., vol. 251, pp.
47-55 (1989); Olsson, R. A., et al., J. Med. Chem., vol. 29, pp.
1683-1689 (1986); Bridges, A. J., et al., J. Med. Chem., vol. 31,
pp. 1282-1285 (1988); Hutchinson, A. J., et al., J. Med. Chem.,
vol. 33, pp. 1919-1924 (1990); Ukena, M., et al., J. Med. Chem.,
vol. 34, pp. 1334-1339 (1991); Francis, J. E., et al., J. Med.
Chem., vol. 34, pp. 2570-2579 (1991); Yoneyama, F., et al., Eur. J.
Pharmacol., vol. 213, pp. 199-204 (1992); Peet, N. P., et al., J.
Med. Chem., vol. 35, pp. 3263-3269 (1992); and Cristalli, G., et
al., J. Med. Chem., vol. 35, pp. 2363-2368 (1992); all of which are
incorporated herein by reference.
[0052] The present method includes the administration of a Type IV
phosphodiesterase (PDE) inhibitor in combination with the compound
of formula (I). Examples of Type IV phosphodiesterase inhibitors
include those disclosed in U.S. Pat. No. 4,193,926, and WO
92-079778, and Molnar-Kimber, K. L., et al., J. Immunol., vol. 150,
p. 295A (1993), all of which are incorporated herein by
reference.
[0053] Specifically, the suitable Type IV phosphodiesterase (PDE)
inhibitors include racemic and optically active
4-(polyalkoxyphenyl)-2-py- rrolidones of general formula (V) 8
[0054] (disclosed and described in U.S. Pat. No. 4,193,926) wherein
R.sup.18 and R.sup.19 each are alike or different and are
hydrocarbon radicals having up to 18 carbon atoms with at least one
being other than methyl, a heterocyclic ring, or alkyl of 1-5
carbon atoms which is substituted by one or more of halogen atoms,
hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino group or
amino.
[0055] Examples of hydrocarbon R.sup.18 and R.sup.19 groups are
saturated and unsaturated, straight-chain and branched alkyl of
1-18, preferably 1-5, carbon atoms, cycloalkyl and cycloalkylalkyl,
preferably of 3-7 carbon atoms, and aryl and aralkyl, preferably of
6-10 carbon atoms, especially monocyclic.
[0056] Examples of alkyl are methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, tert-butyl, pentyl, 2-methylbutyl,
2,2-dimethylpropyl, hexyl, heptyl, octyl, nonyl,
1,2-dimethylheptyl, decyl, undecyl, dodecyl and stearyl, with the
proviso that when one of R.sup.18 and R.sup.19 is methyl, the other
is a value other than methyl. Examples of unsaturated alkyl groups
are alkenyl and alkynyl, e.g., vinyl, 1-propenyl, 2-propenyl,
2-propynyl and 3-methyl-2-propenyl.
[0057] Examples of cycloalkyl and cycloalkylalkyl which preferably
contain a total of 3-7 carbon atoms are cyclopropyl,
cyclopropylmethyl, cyclopentyl and cyclohexyl.
[0058] Examples of aryl and aralkyl are phenyl and benzyl, which
are preferred, and tolyl, xylyl, naphthyl, phenethyl and
3phenylpropyl.
[0059] Examples of heterocyclic R.sup.18 and R.sup.19 groups are
those wherein the heterocyclic ring is saturated with 5 or 6 ring
members and has a single 0, S or N atom as the hetero atom, e.g.,
2- and 3-tetrahydrofuryl, 2 and 3-tetrahydropyranyl, 2- and
3-tetrahydrothiophenyl, pyrrolidino, 2- and 3-pyrrolidyl,
piperidino, 2-, 3- and 4-piperidyl, and the corresponding
N-alkyl-pyrrolidyl and piperidyl wherein alkyl is of 1-4 carbon
atoms. Equivalents are heterocyclic rings having fewer or more,
e.g., 4 and 7, ring members, and one or more additional hetero
atoms as ring members, e.g., morpholino, piperazino and
N-alkylpiperazino.
[0060] Examples of substituted alkyl R.sup.18 and R.sup.19 groups,
preferably of 1-5 carbon atoms, are those mono- or polysubstituted,
for example, by halogen, especially fluorine, chlorine and bromine.
Specific examples of such halogen-substituted alkyl are
2-chloroethyl, 3-chloropropyl, 4-bromobutyl, difluoromethyl,
trifluoromethyl, 1,1,2-trifluoro-2-chloroethyl,
3,3,3-trifluoropropyl, 2,2,3,3,3-pentafluoropropyl and
1,1,1,3,3,3-hexafluoro-2-propyl. Examples of other suitable
substituents for such alkyl groups are hydroxy groups, e.g.,
2-hydroxyethyl or 3-hydroxypropyl; carboxy groups, e.g.,
carboxymethyl or carboxyethyl; alkoxy groups, wherein each alkoxy
group contains 1-5 carbon atoms, e.g., ethoxymethyl,
isopropoxymethyl, 2-methoxyethyl, 2-isopropoxyethyl,
2-butyoxyethyl, 2-isobutyoxyethyl, and 3-pentoxypropyl.
[0061] Also suitable as preferably terminal-positioned substituents
on alkyl groups of 1-5 carbon atoms are alkoxycarbonyl of 1-5
carbon atoms in the alkoxy group. Examples of such alkoxycarbonyl
substituted alkyl-groups are ethoxycarbonylmethyl and
2-butoxycarbonylethyl.
[0062] Alkyl groups of 1-5 carbon atoms can also be substituted,
e.g., in the .beta., .UPSILON. and preferably terminal position
with amino groups wherein the nitrogen atom optionally is mono- or
disubstituted by alkyl, preferably of 1-5 carbon atoms, or is part
of a 4- to 7-membered ring.
[0063] Rolipram and its analogues are specific examples of
preferred Type IV phosphodiesterase inhibitors.
[0064] Examples of inflammatory diseases which may be treated
according to the present invention include:
[0065] autoimmune diseases such as lupus erythenatosis, multiple
sclerosis, type I diabetes mellits, Crohn's disease, ulcerative
colitis, inflammatory bowel disease, osteoporosis, arthritis,
allergic diseases such as asthma, infectious diseases such as
sepsis, septic shock, infectious arthritis, endotoxic shock, gram
negative shock, toxic shock,cerebral malaria, bacterial meningitis,
adult respiratory distress syndrome (ARDS), TNF.alpha.-enhanced HIV
replication and TNF.alpha. inhibition of reverse transciptase
inhibitor activity, wasting diseases (cachexia secondary to cancer
and HIV), skin diseases like psoriasis, contact dermatitis, eczema,
infectious skin ulcers, cellulitis, organ transplant rejection
(including bone marrow, kidney, liver, lung, heart, skin
rejection), graft versus host disease, adverse effects from
amphotericin B treatment, adverse effects from interleukin-2
treatment, adverse effects from OKT3 treatment, adverse effects
from GM-CSF treatment, adverse effects of cyclosporine treatment
and adverse effects of aminoglycoside treatment, ischemia,
mucositis, infertility from endometriosis, circulatory diseases
induced or exacerbated by an inflammatory response such as
atherosclerosis, peripheral vascular disease, restenosis following
angioplasty, inflammatory aortic aneurysm, ischemia/reperfusion
damage, vasculitis, stroke, congestive heart failure, hemorrhagic
shock, vasospasm following subarachnoid hemorrhage, vasospasm
following cerebrovascular accident, pleuritis, pericarditis, and
encephalitis.
[0066] The exact dosage of the compound of formula (I) to be
administered will, of course, depend on the size and condition of
the patient being treated, the exact condition being treated, and
the identity of the particular compound of formula (I) being
administered. However, a suitable dosage of the compound of formula
(I) is 0.5 to 100 .mu.g/kg of body weight, preferably 1 to 10
.mu.g/kg of body weight. Typically, the compound of formula (I)
will be administered from 1 to 8, preferably 1 to 4, times per
day.
[0067] The preferred mode of administration of the compound of
formula (I) may also depend on the exact condition being treated.
However, most typically, the mode of administration will be oral,
topical, intravenous, parenteral, subcutaneous, or intramuscular
injection.
[0068] Of course, it is to be understood that the compound of
formula (I) may be administered in the form of a pharmaceutically
acceptable salt. Examples of such salts include acid addition
salts. Preferred pharmaceutically acceptable addition salts include
salts of mineral acids, for example, hydrochloric acid, sulfuric
acid, nitric acid, and the like; salts of monobasic carboxylic
acids, such as, for example, acetic acid, propionic acid, and the
like; salts of dibasic carboxylic acids, such as maleic acid,
fumaric acid, oxalic acid, and the like; and salts of tribasic
carboxylic acids, such as, carboxysuccinic acid, citric acid, and
the like. In the compounds of formula (I) in which R is
--CO.sub.2H, the salt may be derived by replacing the acidic proton
of the --CO.sub.2H group with a cation such as Na.sup.+, K.sup.+,
NH.sup.+.sub.4 mon-, di, tri, or tetra(C.sub.1-4-alkyl)ammonium, or
mono-, di-, tri-, or tetra(C.sub.2-4alkanol)ammonium.
[0069] It is also to be understood that many of the compounds of
formula (I) may exist as various isomers, enantiomers, and
diastereomers and that the present invention encompasses the
administration of a single isomer, enantiomer, or diastereomer in
addition to the administration of mixtures of isomers, enantiomers,
or diastereomers.
[0070] The compounds of formula (I) can be administered orally, for
example, with an inert diluent with an edible carrier. They can be
enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the compounds can be
incorporated with excipients and used in the form of tablets,
troches, capsules, elixirs, suspensions, syrups, waters, chewing
gums, and the like. These preparations should contain at least 0.5%
by weight of the compound of formula (I), but the amount can be
varied depending upon the particular form and can conveniently be
between 4.0% to about 70% by weight of the unit dosage. The amount
of the compound of formula (I) in such compositions is such that a
suitable dosage will be obtained. Preferred compositions and
preparations according to the present invention are prepared so
that an oral dosage unit form contains between about 30 .mu.g and
about 5 mg, preferably between 50 to 500 .mu.g, of active
compound.
[0071] Tablets, pills, capsules, troches, and the like can contain
the following ingredients: a binder, such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient, such as starch
or lactose; a disintegrating agent, such as alginic acid, Primogel,
corn starch, and the like; a lubricant, such as magnesium stearate
or Sterotes; a glidant, such as colloidal silicon dioxide; a
sweetening agent, such as sucrose, saccharin or aspartame; or
flavoring agent, such as peppermint, methyl salicylate, or orange
flavoring. When the dosage unit form is a capsule it can contain,
in addition to the compound of formula (I), a liquid carrier, such
as a fatty oil.
[0072] Other dosage unit forms can contain other materials that
modify the physical form of the dosage unit, for example, as
coatings. Thus, tablets or pills can be coated with sugar, shellac,
or other enteric coating agents. A syrup may contain, in addition
to the active compounds, sucrose as a sweetening agent and
preservatives, dyes, colorings, and flavors. Materials used in
preparing these compositions should be pharmaceutically pure and
non-toxic in the amounts used.
[0073] For purposes of parenteral therapeutic administration, the
compounds of formula (I) can be incorporated into a solution or
suspension. These preparations should contain at least 0.1% of the
aforesaid compound, but may be varied between 0.5% and about 50% of
the weight thereof. The amount of active compound in such
compositions is such that a suitable dosage will be obtained.
Preferred compositions and preparations according to the present
invention are prepared so that a parenteral dosage unit contains
between 30 .mu.g to 5 mg, preferably between 50 to 500 .mu.g, of
the compound of formula (I).
[0074] Solutions or suspensions of the compounds of formula (I) can
also include the following components: a sterile diluent, such as
water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine, propylene glycol or other synthetic solvents:
antibacterial agents, such as benzyl alcohol or methyl parabens;
antioxidants, such as ascorbic acid or sodium bisulfite; chelating
agents, such as ethylenediaminetetraacetic acid; buffers, such as
acetates, citrates or phosphates; and agents for the adjustment of
tonicity, such as sodium chloride or dextrose. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
[0075] Effective amounts of the Type IV phosphodiesterase inhibitor
can be administered to a subject by any one of various methods, for
example, orally as in a capsule or tablets, topically, or
parenterally in the form of sterile solutions. The Type IV
phosphodiesterase inhibitors, while effective themselves, can be
formulated and administered in the form of their pharmaceutically
acceptable addition salts for purposes of stability, convenience of
crystallization, increased solubility, and the like.
[0076] Preferred pharmaceutically acceptable addition salts include
salts of mineral acids, for example, hydrochloric acid, sulfuric
acid, nitric acid, and the like; salts of monobasic carboxylic
acids, such as, for example, acetic acid, propionic acid, and the
like; salts of dibasic carboxylic acids, such as maleic acid,
fumaric acid, oxalic acid, and the like; and salts of tribasic
carboxylic acids, such as, carboxysuccinic acid, citric acid, and
the like.
[0077] The Type IV phosphodiesterase may be administered in the
form of a pharmaceutical composition similar to those described
above in the context of the compound of formula (I).
[0078] While dosage values will vary with the specific disease
condition to be alleviated, good results are achieved when the Type
IV phosphodiesterase inhibitor is administered to a subject
requiring such treatment as an effective oral, parenteral or
intravenous dose as described below.
[0079] For oral administration, the amount of active agent per oral
dosage unit usually is 0.1-20 mg, preferably 0.5-10 mg. The daily
dosage is usually 0.1-50 mg, preferably 1-30 mg. p.o. For
parenteral application, the amount of active agent per dosage unit
is usually 0.005-10 mg, preferably 0.01-5 mg. The daily dosage is
usually 0.01-20 mg, preferably 0.02-5 mg i.v. or i.m.
[0080] With topical administration, dosage levels and their related
procedures would be consistent with those known in the art, such as
those dosage levels and procedures described in U.S. Pat. No.
5,565,462 to Eitan et al., which is incorporated herein by
reference.
[0081] It is to be understood, however, that for any particular
subject, specific dosage regimens should be adjusted to the
individual need and the professional judgement of the person
administering or supervising the administration of the Type IV
phosphodiesterase inhibitor. It is to be further understood that
the dosages set forth herein are exemplary only and that they do
not, to any extent, limit the scope or practice of the present
invention.
[0082] In a particularly preferred embodiment, the compound of
formula (I) and the Type IV phosphodiesterase inhibitor are
coadministered together in a single dosage unit. The compound of
formula (I) and the type IV phosphodiesterase inhibitor may be
administered in the same type of pharmaceutical composition as
those described above in the context of the compound of formula
(I).
[0083] By coadministering a Type IV phosphodiesterase inhibitor
with the agonist of the A.sub.2A adenosine receptor it is possible
to dramatically lower the dosage of the A.sub.2A adenosine receptor
agonist and the Type IV phosphodiesterase inhibitor due to a
synergistic effect of the two agents. Thus, in the embodiment
involving coadministration of the A.sub.2A adenosine receptor
agonist with the type IV phosphodiesterase inhibitor, the dosage of
the A.sub.2A adenosine receptor agonist may be reduced by a factor
of 5 to 10 from the dosage used when no type IV phosphodiesterase
inhibitor is administered. This reduces the possibility of side
effects.
[0084] The present invention will now be described in more detail
in the context of the coadministration of WRC-0470, WRC-0474[SHA
211], WRC-0090 or WRC-0018 and rolipram. However, it is to be
understood that the present invention may be practiced with other
compounds of formula (I) and other Type IV phosphodiesterase
inhibitors of formula (V).
[0085] The present studies establish that anti-inflammatory doses
have no toxic effects in animals; the effect of WRC-0470 to inhibit
neutrophil activation is synergistic with rolipram; and intravenous
infusion of WRC-0470 profoundly inhibits extravasation of
neutrophils in an animal model of inflammation, an action also
synergistic with rolipram. Further, the present studies establish
that activation of A.sub.2A receptors on human monocytes strongly
inhibits TNF.alpha. (an inflamatory cytokine) release. This
mechanism further contributes to the anti-inflamatory action of the
A.sub.2A adenosine receptor agonists of the present invention.
[0086] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLES
[0087] Materials and Methods
[0088] Materials: f-Met--Leu--Phe(fMLP), luminol, and trypan blue
were from Sigma Chemical. Ficoll-hypaque was purchased from Flow
Laboratories (McLean, A) and Los Alamos Diagnostics (Los Alamos, N.
Mex.). Hanks balanced salt solution (HBSS), and limulus amebocyte
lysate assay kit were from Whittaker Bioproducts (Walkersville,
Md.). Human serum albumin (HSA) was from Cutter Biological
(Elkhart, Ind.). Recombinant human tumor necrosis factor-alpha was
supplied by Dianippon Pharmaceutical Co. Ltd. (Osaka, Japan).
ZM241385 was a gidt of Dr. Simon Poucher, Zeneca Pharmaceuticals
(Chesire, England).
[0089] Leukocyte Preparation: Purified PMN (.about.98% PMN and
>95% viable by trypan blue exclusion) containing <1 platelet
per 5 PMN and <50 pg/ml endotoxin (limulus amebocyte lysate
assay) were obtained from normal heparinized (10 Units/ml) venous
blood by a one step ficoll-hypaque separation procedure (Ferrante,
A., et al., J. Immunol. Meth., vol. 36, p. 109, (1980)). Residual
RBC were lysed by hypotonic lysis with iced 3 ml 0.22% sodium
chloride solution for 45 seconds followed by 0.88 ml of 3% sodium
chloride solution.
[0090] Chemiluminescence: Luminol enhanced chemiluminescence, a
measure of neutrophil oxidative activity, is dependent upon both
superoxide production and mobilization of the granule enzyme
myeloperoxidase. The light is emitted from unstable high-energy
oxygen species generated by activated neutrophils. Purified PMN
(5.times.10.sup.5/ml) were incubated in HBSS containing 0.1 % human
serum albumin (1 ml) with or without adenosine, adenosine analogs,
and TNF.alpha. (1 U/mL) for 30 minutes at 37.degree. C. in a
shaking water bath. Then luminol (1.times.10.sup.-4M) enhanced
f-met-leu-phe (1 .mu.M) stimulated chemiluminescence was read with
a Chronolog Photometer (Chrono-log Corp., Havertown, Pa.) at
37.degree. C. for 8 min. Chemiluminescence is reported as relative
peak light emitted (=height of the curve) compared to samples with
TNF and without adenosine or adenosine analogs. WRC-0474[SHA 211]
was 10 times more potent than either adenosine (ADO) or CGS21680 in
decrease TNF.alpha.-primed f-met-leu-phe-stimulated PMN
chemiluminescence (see FIG. 1).
[0091] Synergy of A.sub.2A Adenosine Receptor Agonist and
Phosphodiesterase Inhibitors. The synergy between WRC-0474[SHA 211]
and 4-(3-cyclopentyloxy-4methoxyphenyl)-2-pyrrolidone (rolipram)
was examined by measuring the effect of combined WRC-0474[SHA 211]
and rolipram on TNF-primed f-met-leu-phe-stimulated suspended
neutrophil superoxide release and on the oxidative burst of
neutrophils adhering to matrix proteins (in this model the PMN
oxidative burst is enhanced by small concentrations of TNF.alpha.
[e.g. 1 U/ml] when added prior to the addition of a second stimulus
such as the peptide f-met-leu-phe).
[0092] Suspended PMN Superoxide Release: Human PMN
(1.times.10.sup.6/ml) from Ficoll-Hypaque separation were primed
for 30 minutes (37.degree. C.) with or without rhTNF (10 U/ml),
with adenosine deaminase (1 U/ml), and with or without
4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone and SHA 211.
Cytochrome c (120 .mu.M), catalase (0.062 mg/ml) and fMLP (100 nM)
were added and the samples incubated for 10 minutes more at
37.degree. C. SOD (200 U/ml) was added to matched samples. The
samples were iced and centrifuged (2000 g.times.10 minutes). The
optical density of the supernatants were read at 550 nm against the
matched SOD samples, and the moles of SOD-inhibitable superoxide
released in 10 minutes were calculated.
[0093] A synergistic effect of WRC-0474[SHA 211] and rolipram in
decreasing the TNF.alpha.-primed fMLP-stimulated PMN oxidative
burst was observed (see FIG. 2).
[0094] TNF.alpha.-stimulated superoxide release of PMN adherent to
a matrix protein (fibrinogen) coated surface: Human PMN
(1.times.10.sup.6/ml) from Ficoll-Hypaque separation were incubated
for 90 minutes in 1 ml of Hanks balanced salt solution containing
0.1% human serum albumin, cytochrome c (120 .mu.M), and catalase
(0.062 mg/ml) in the presence and absence of rhTNF (1 U/ml),
WRC-0474[SHA 211] (10 nM) and rolipram (100 nM) in a tissue culture
well which had been coated overnight with human fibrinogen. SOD
(200 U/ml) was added to matched samples. The supernatants were iced
and centrifuged (2000 g.times.10 minutes) to remove any remaining
suspended cells, and the optical density of the supernatants were
read at 550 mn against the matched SOD samples, and the nmoles of
SOD-inhibitable superoxide released in 90 minutes were
calculated.
[0095] A synergistic effect of WRC-0474[SHA 211] and rolipram in
decreasing the TNF.alpha.-stimulated release of superoxide from PMN
adherent to fibrinogen was observed (see FIG. 3).
[0096] Effect of WRC-0474[SHA 211] with and without rolipram on
TNF-Stimulated PMN Adherence to a Fibrinogen-Coated Surface.
Cronstein et al., J. Immunol., vol. 148, p. 2201 (1992) reported
that adenosine binding to A.sub.1 receptors increases PMN adherence
to endothelium and matrix proteins and binding to A.sub.2 receptors
decreases adherence to these surfaces when the PMN are stimulated
with fMLP. Despite this, others have failed to see much of an
effect of adenosine (10 .mu.M) on TNF.alpha.-stimulated PMN
adherence to matrix proteins. In contrast, adenosine dramatically
decreases the oxidative burst of TNF.alpha.-stimulated PMN adhering
to matrix proteins (DeLa Harpe, J., J. Immunol., vol. 143, p. 596
(1989)). The experiments described above establish that
WRC-0474[SHA 211] decreases TNF-stimulated oxidative activity of
PMN adhering to fibrinogen, especially when combined with
rolipram.
[0097] PMN adherence to fibrinogen was measured as follows as
adapted from Hanlon, J. Leukocyte Biol., vol. 50, p. 43 (1991).
Twenty-four well flat-bottomed tissue culture plates were incubated
(37.degree. C.) overnight with 0.5 ml of fibrinogen (5 mg/ml)
dissolved in 1.5% NaHCO.sub.3. The plates were emptied and each
well washed 2.times. with 1 ml of normal saline. The wells were
then filled with 1 ml of HBSS-0. 1% human serum albumin containing
PMN (1.times.10.sup.6/ml) with and without rhTNF.alpha.(1 U/ml),
adenosine deaminase (ADA) (1 U/ML), WRC-0474[SHA 211] (10 nM),
CGS21680 (30 nM), adenosine (100 nM) and rolipram (100 nM). The
plates were incubated for 90 minutes at 37.degree. C. in 5%
CO.sub.2. Following incubation the tissue culture wells were washed
free of non-adherent cells with normal saline. The adherent
monolayer of PMN was lysed with 0.1% triton-X, the amount of lactic
dehydrogenase (LDH) released from the monolayer assayed (LDH kit,
Sigma Co., St. Louis, Mo.), and compared to a standard curve
relating the LDH content to PMN numbers. The results are shown in
FIG. 4.
[0098] As a comparison to WRC-0474[SHA 211] (at only 10 nM),
CGS21680 (30 nM) decreased TNF-stimulated adherence in the presence
of ADA from 38% to 30% adhered (p=0.004) (see FIG. 4), and ten
times as much adenosine (100 nM) decreased adherence to 28% adhered
(p=0.009 compared to TNF in the presence of ADA).
[0099] Additional effects of adenosine A.sub.2A agonists on
adherent human neutrophil oxidative activity. The bioactivity of
test compounds WRC-0474[SHA 211], WRC-0470, WRC-0090 and WRC-0018
were evaluated according to the following method modified from
Sullivan, G. W. et al., Int. J. Immunonopharmacol, 1995,
17:793-803. Neutrophils (1.times.10.sup.6/ml) from Ficoll-Hypaque
separation were incubated for 90 minutes in 1 ml of Hanks balanced
salt solution containing 0.1% human serum albumin, cytochrome c
(120 .mu.M) and catalase (0.062 mg/ml) in the presence and absence
of rhTNF.alpha. (1 U/ml), WRC-0474[SHA 211], WRC-0470, WRC-0090 and
WRC-0018 (3-300 nM), and rolipram (100 nM) in a tissue culture well
which had been coated overnight with human fibrinogen. The
supernatants were iced and centrifuged (200 g.times.10 min) to
remove any remaining suspended cells, and the optical densities of
the supernatants were read at 550 nm against matched superoxide
dismutase (SOD) (200 U/ml) samples. The nmoles of SOD=inhabitable
superoxide released in 90 min were calculated.
[0100] FIG. 5 shows synergy between A.sub.2A adenosine agonists and
rolipram in inhibiting TNF.alpha.-stimulated adherent PMN oxidative
activity (p<0.05). WRC-0474[SHA 211] (30-300 nM), WRC-0470 (300
nM), WRC-0090 (300 nM) and WRC-0018 (300 nM) combined with rolipram
synergistically decreased superoxide release (p<0.05). All four
compounds had some activity in the presence of rolipram.
WRC-0474[SHA 211] and WRC-0470 were the most active. Nanomolar
concentrations of WRC-0474[SHA 211] resulted in biphasic activity.
All compounds were synergistic with rolipram to decrease
TNF.alpha.-stimulated adherent PMN oxidative activity.
[0101] PMN degranulation (adherent cells). The following methods
were adapted from Sullivan, G. W. and G. L. Mandell, Infect.
Inumun., 1980: 30:272-280. Neutrophils (3.1.times.10.sup.6/ml) from
Ficoll-Hypaque separation were incubated for 120 minutes in 1 ml of
Hanks balanced salt solution containing 0.1% human serum albumin,
.+-.rh TNF.alpha. (10 U/ml), .+-.WRC-0470 (3-300 nM), and
.+-.rolipram (300 nM) in a tissue culture well which had been
coated overnight with human fibrinogen. The supernatant fluids with
any suspended neutrophils were harvested following incubation,
centrifuged (2000.times.g for 10 min) to remove any suspended cells
and the cell-free supernatants frozen. Release of lysozyme, a
component of neutrophil primary and secondary granules was assayed.
Lysis of a suspension of Micrococcus lysodeikticus by the
"cell-free supernatant" was measured by spectrophotometric analysis
(540 mm) to determine the amount of release of granule contents to
the surrounding medium.
[0102] Results showed that WRC-0470 (300 nM) with rolipram (300 nM)
significantly decreased TNF.alpha.-stimulated adherent neutrophil
degranulation 67%; P=0.027. The data indicate that in addition to
decreasing TNF.alpha.-stimulated PMN adherent and the oxidative
burst of these adherent neutrophils, WRC-0470 also decreases
degranulation activated PMN adhering to a biological surface.
[0103] PMN oxidative activity in whole blood. The following methods
were adapted from Rothe, G. A. et al., J. Immunol. Meth. 1991;
138:133-135). Heparinized whole blood (0.8 ml) was incubated
(37.degree.; 30 min) with adenosine deaminase (ADA, 1 U/ml),
catalase (14,000 U/ml), .+-.dihydrorhodamine 123, .+-.WRC-0470
(3-300 nM), .+-.rolipram (300 nM) and .+-.TNF.alpha. (10 U/ml). The
primed blood samples were stimulated with fMLP (15 min), then iced,
the red blood cells lysed with FACS lysing solution
(Becton-Dickinson, San Jose, Calif.), washed and the leukocytes
resuspended in phosphate buffered saline (PBS) These samples
containing mixed leukocytes were gated for neutrophils by forward
and side scatter and the fluorescence of 10,000 neutrophils
measured in the FL1 channel of a FACScan (Beckton-Dickinson)
fluorescence activated cell sorter.
[0104] The results are reported as relative mean florescence
intensity in FIG. 6 of the drawings. WRC-0470 decreased oxidative
activity of TNF.alpha.-primed fMLP-stimulated neutrophils in whole
blood and acted synergistically with rolipram. WRC-0470 (30-300 nM)
decreased neutrophil oxidative activity synergistically with
rolipram (300 nM) in samples stimulated with fMLP and in blood
samples primed with TNF.alpha. and then stimulated with fMLP.
[0105] Production of TNF.alpha. by purified human adherent
monocytes. A monocyte rich monolayer (>95% monocytes) was
prepared by incubating 1 ml of the mononuclear leukocyte fraction
(5.times.10.sup.5/ml) from a Ficoll-Hypaque separation in wells of
a 24 well tissue culture plate (1 hr; 37.degree. C.; 5% CO.sub.2).
The non-adherent leukocytes were removed by washing and culture
medium added to the wells (1 ml RPMI 1640 containing 1.5 mM
HEPES-1% autologous serum with penicillin and streptomycin (250
U/ml and 250 .mu.g/ml, respectively) and ADA (1 U/ml) .+-.WRC-0470
(30-100 nM), .+-.endotoxin (10 ng/ml), +rolipram (300 nM) and
.+-.the adenosine A.sub.2A selective antagonist
4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3a]-[1,3,5]trazinyl-amino]eth-
yl)-phenol (ZM241385) (50 nM). The samples were incubated for 4 hr
(37.degree. C.; 5% CO.sub.2) and the supernatants harvested. Any
suspended cells were removed by centrifugation and the cell-free
samples frozen (-70.degree. C.). TNF.alpha. was assayed in the
cell-free supernatants by an ELISA kit (Cistron Biotechnology, Pine
Brook, N.J.).
[0106] As shown in FIGS. 7A and 7B, WRC-0470 .+-.rolipram-decreased
endotoxin-stimulated adherent monocyte production of TNF.alpha.
(P<0.050). As illustrated in FIG. 7B, the A.sub.2A selective
antagonist ZM241385 significantly inhibited the effect of WRC-0470
(300 nM) combined with rolipram (300 nM) (p=0.020) on TNF.alpha.
release from monocytes. Hence, WRC-0470 affects
TNF.alpha.-stimulated neutrophil activity and decreases
endotoxin-stimulated TNF.alpha. production by monocytes.
[0107] Effects of WRC-0470 and rolipram on the extravasation of
white blood cells in a rat model of inflammation. Adult wistar rats
(approximately 200 g) were anesthetized with intermuscular
injections of ketamine and xylazine. Bacteria meningitis (BM) was
induced via intracisternal inoculation of either E. coli strain
026:B6LPS (200 ng), cytokines (IL-1 and TNF.alpha., or LPS plus
cytokines). The animals were then infused with rolipram and/or
WRC-1470 over the duration of the experiment using a Harvard pump.
CSF (cerebrospinal fluid) and blood was then sampled at 4 h
postinoculation and alterations in BBBP (blood-brain barrier
permeability) and WBC (white blood cell) counts were determined.
CSF and WBC concentrations were determined with standard
hemacytometer methods. For assessment of % BBBP, rats were given an
intravenous injection of 5 .mu.Ci 125I-labeled bovine serum albumin
concomitant with intracisternal inoculation. Equal samples of CSF
and blood were read simultaneously in a gamma counter and after
subtraction of background radioactivity, % BBBP was calculated by
the following formula: % BBBP=(cpm CSF/cpm blood).times.100. All
statistical tests were performed using Instat biostatistical
software to compare the post-inoculation samples of experimental
rats with the control rats. The statistical tests used to generate
p-values were Student's t-test and ANOVA.
[0108] Results of the tests are reported in FIGS. 8 and 9. Infusion
of WRC-0470 at a rate of 0.005-1.2 .mu.g/kg/hr inhibited
pleiocytosis (p<0.05 as compared to control). The effect of
WRC-0470 on BBBP is shown in FIG. 9. A significant response is seen
with a range of 0.01-0.015 .mu.g/kg/hr (p<0.05 as compared to
control). A rebound effect is noted with the administration of 1.2
.mu.g/kg/hr where % BBBP returned to control. FIG. 10 shows the
effect of rolipram on CSF pleocytosis in a range of 0-0.01
.mu.g/kg/hr with 0.01 .mu.g/kg/hr inhibiting 99% of the pleocytosis
(p<0.05). Rolipram at either 0.01 or 0.005 .mu.g/kg/hr showed
significant inhibition of alterations of BBBP (p<0.05), while a
dose of 0.002 .mu.g/kg/hr had no significant effect
[0109] The effect of a combination of rolipram and WRC-0470 on CSF
WBC pleocytosis is illustrated in FIG. 11. Rolipram (0.001
.mu.g/kg/hr) in combination with WRC-0470 (0.1 .mu.g/kg/hr)
inhibited migration of WBC's (200.+-.70 WBC/.mu.l) into the
sub-arachnoid space (SAS) to a greater extent than did either
rolipram (1,670.+-.1,273 WBC/.mu.l, p<0.050) or WRC-0470
(600.+-.308 WBCs/.mu.l, p<0.050) alone. The data show a powerful
inhibiting effect of WRC-0470 and a synergy with rolipram to
prevent inflammation in an animal model.
[0110] Application of A.sub.2A adenosine receptors with or without
rolipram on balloon angioplasty and gene therapy. Balloon
angioplasty is commonly used to treat coronary artery stenosis.
Restenosis following balloon angioplasty (BA) occurs in up to 40%
of coronary interventions. Holmes et al., American Journal of
Cardiology, 53: 77C-81C (1984). (40%). Restenosis results from a
complex interaction of biologic processes, including (i) formation
of platelet-rich thrombus; (ii) release of vasoactive and mitogenic
factors causing migration and proliferation of smooth muscle cells
(SMC); (iii) macrophage and other inflammatory cell accumulation
and foam cell (FC) formation; (iv) production of extracellular
matrix; and (v) geometric remodeling. Recently the use of coronary
stents and pharmacologic intervention using a chimeric antibody to
block the integrin on platelets have been partially successful in
limiting restenosis after percutaneous coronary interventions in
man. Topol et al., Lencet, 343: 881-886 (1994). Since inflammatory
cell infiltration might be central to the response to injury, and
restenotic processes, and adenosine, activing via A.sub.2A
adenosine receptors, inhibits tissues inflammatory cell
accumulation, we hypothesize that agonists of A.sub.2A adenosine
receptors.+-.type IV PDE inhibitors will reduce the incidence of
restenosis following balloon angioplasty.
[0111] In addition, recent advances in local delivery catheters and
gene delivery techniques raise the interesting and exciting
possibility of administering genes locally into the vessel wall.
Nabel et al., Science, 249: 1285-1288 (1990); Leclerc et al.,
Journal of Clinical Investigation, 90: 936-944 (1992).
Adenoviral-mediated gene transfer affords several advantages over
other techniques. However, gene expression is only transient, and
has been observed for 7-14 days with diminuition or loss of
expression by 28 days. Lack of persistence may result from host
immune cytolytic responses directed against infected cells. The
inflammatory response generated by the present generation of
adenovirus results in neoitimal lesion formation and may thus
offset the benefit of a therapeutic gene. Newman et al., Journal of
Clinical Investigation, 96: 2955-2965 (1995). An A.sub.2A adenosine
receptor agonist.+-.a type IV phosphodiesterase inhibitor in
combination with adenovirus may improve the efficiency of gene
transfer.
[0112] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
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